Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.21.1 (DNase)
7,655 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A deoxyribonuclease has been purified more than 2000-fold from the green algae, Chlamydomonas reinhardii. The enzyme is most active on denatured DNA. Optimum activity is at pH 8.5, in 80 mM Tris-HCl buffer and 2 mM CaCl2. Other divalent cations can replace Ca2+ with varying lower efficiency. EDTA and inorganic phosphate are strongly inhibitory, while ATP and high concentrations of 2-mercaptoethanol are slightly inhibitory. The molecular weight is approximately 35 000, the Stokes radius is 2.7 nm, and the sedimentation coefficient 2.8 S. It is a single polypeptide chain, and the frictional ratio of 1.27 suggests it is only slightly asymetrical. The isoelectric point is 9.5. This enzyme has been termed exonuclease 1.
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PMID:A deoxyribonuclease from Chlamydomonas reinhardii. 1. Purification and properties. 1 43

Chlamydomonas reinhardtii cells treated with toluene at 0 degrees C and 25 degrees C incorporate ribonucleoside triphosphates (NTPs) into chloroplast RNA at 25 degrees C and also at 35 degrees C. The incorporation requires all four NTPs and Mg2+, and is completely inhibited by DNase, RNase, actinomycin D (40 microgram/ml) and rifampicin (350 microgram/ml). However, the incorporation is almost totally insensitive to both alpha-amanitin and streptolydigin at 200 microgram/ml.
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PMID:Synthesis of chloroplast ribonucleic acid in Chlamydomonas reinhardtii toluene-treated cells. 25 50

We have found a unique deoxyribonuclease in extracts of the eukaryotic green alga Chlamydomonas. When incubated with viral DNA from adenovirus-2, this enzyme produces discrete fragments that form bands upon electrophoresis in an agarose gel. Site specificity of the enzymatic cleavage examined by identifying the 5'-terminal nucleotides in cleaved adenovirus-2 DNA and by studies with synthetic polynucleotides of defined sequence, indicates that the initial endonucleolytic cleavage occurs at a site containing a deoxythymidine residue. Electron microscopy of cleaved adenovirus-2 DNA revealed single-strand segments within duplex DNA. We propose that the enzyme acts by making initial site-specific single-strand incisions, followed by subsequent excision on the same strand, producing a gapped duplex molecule; and that double-strand scissions result from limited occurrence of overlapping single-strand gaps on complementary strands.
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PMID:A site-specific single-strand endonuclease from the eukaryote Chlamydomonas. 26 18

The properties of three DNA polymerase species A, B and C, purified from Chlamydomonas reinhardii were compared. DNA polymerases A and B have Km values with respect to deoxyribonucleoside triphosphates of 19 micron and 3 micron respectively. DNA polymerase A is most active with activated DNA, but will also use native DNA and synthetic RNA and DNA templates with DNA primers. DNA polymerase B is also most active with activated DNA, but will use denatured DNA and synthetic DNA templates. It is inactive with RNA templates. DNA polymerase B is completely inactive in the presence of 100 micron-heparin, which has no effect on DNA polymerase A activity. Heparin dissociates DNA polymerase B into subunits that are still catalytically active, but which heparin inhibited. DNA polymerase B possesses deoxyribonuclease activity that is inhibited by 5 micron-heparin, suggesting that the deoxyribonuclease is an integral part of the DNA polymerase moiety. DNA polymerase A is devoid of nuclease activity. DNA polymerase C is similar to DNA polymerase B in all these properties, though it is more active with RNA primers and has greater heat-sensitivity.
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PMID:DNA polymerases from Chlamydomonas reinhardii. Further characterization, action of inhibitors and associated nuclease activities. 64 18

A deoxyribonuclease purified Chlamydomonas reinhardii has been shown to be specific for single-stranded DNA. The enzyme is most active on thermally denatured DNA, but also degrades single-stranded termini from double-stranded DNA. The enzyme has no effect on single-stranded or double-stranded intact circular phiX174DNA, suggesting that it requires DNA termini for activity. DNA is digested progressively to oligonucleotides and then mononucleotides. The product of the reaction is nucleoside 5'-monophosphates. The enzyme has no effect on RNA, nor does it possess phosphatase or phosphodiesterase activity. No specificity was demonstrated for phosphate or hydroxyl groups at either the 5' or 3' termini of DNA. The enzyme may be able to initiate hydrolysis at either the 3' or the 5' termini, since radioactivity was released more rapidly from 5' and 3' termini than from bulk DNA. The enzyme has been tentatively named Chlamydomonas reinhardii exonuclease 1.
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PMID:A deoxyribonuclease from Chlamydomonas reinhardii. 2. Substrate specificity, mode of action and products. 88 35

All chloroplast 23S ribosomal RNA genes of the unicellular alga Chlamydomonas reinhardtii contain an 888 bp group I intron with an internal open reading frame (ORF). A precursor RNA encompassing the intron with its 5' and 3' flanking sequences was shown to self-splice both during in vitro transcription and upon incubation of the isolated pre-RNA under self-splicing conditions. Expression of the internal ORF in Escherichia coli in the presence of a plasmid containing a cDNA corresponding to the intronless form of the 23S rRNA gene resulted in specific cleavage of the cDNA at or close to the exon junction sequence. To test whether this ORF-encoded double-strand DNA endonuclease is involved in intron mobility in vivo, the same ribosomal cDNA was stably integrated into the C. reinhardtii chloroplast genome using particle gun mediated transformation. All the transformants with the cDNA integrated at the expected site in the chloroplast genome had the intron precisely inserted at the artificial exon junction site. These experiments demonstrate that the chloroplast ribosomal intron of C. reinhardtii behaves as a ribozyme in vitro and also as a mobile genetic element in vivo provided a target site is present.
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PMID:Chloroplast ribosomal intron of Chlamydomonas reinhardtii: in vitro self-splicing, DNA endonuclease activity and in vivo mobility. 191 4

During interspecific crosses between Chlamydomonas eugametos and Chlamydomonas moewusii, an optional group I intron of 955 base pairs (CeLSU.5) in the C. eugametos chloroplast large subunit rRNA gene undergoes a duplicative transposition event which is associated with frequent co-conversion of flanking cpDNA sequences. In the present study, we show that the basic protein of 218 amino acids encoded by CeLSU.5 could mediate the phenomenon of intron transposition, also called intron homing. We overexpressed the ORF specifying this protein in E. coli using expression vectors that contain a C. moewusii cpDNA sequence encompassing the intron homing site. The expression product was found to exhibit a double-strand DNA endonuclease activity that is specific for the homing site. This activity was detected in vivo by self-linearization of the expression plasmids.
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PMID:A group I intron in the chloroplast large subunit rRNA gene of Chlamydomonas eugametos encodes a double-strand endonuclease that cleaves the homing site of this intron. 203 85

We show that in E. coli, a Chlamydomonas chloroplast promoter, PA, is repressed by Integration Host Factor (IHF). The himA 42 mutation, altering the alpha-subunit of E. coli IHF, leads to over-accumulation of PA transcripts in vivo. This effect requires upstream chloroplast DNA sequences. DNAase I and methylation protection experiments show that IHF binds in vitro to a site within PA and band-retardation shows that IHF inhibits formation of PA-E. coli RNA polymerase open complexes. We interpret these results, together with our previous deletion analyses, to mean that in E. coli, repression of PA by IHF minimally requires both binding of IHF to a site overlapping PA and binding of one or more additional proteins, perhaps including IHF itself, to sequences upstream of PA.
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PMID:Integration host factor (IHF) represses a Chlamydomonas chloroplast promoter in E. coli. 328 26

1. The activities of DNA polymerase preparations from the algae Euglena gracilis, Chlamydomonas reinhardtii, Chlorella pyrenoidosa, Anabaena variabilis and Anacystis nidulans were measured. The blue-green algae Anabaena and Anacystis contain a 5-20-fold higher activity of the enzyme than do the green algae. DNA polymerases from the blue-green algae show a pH optimum of 9 and prefer a relatively low Mg(2+) concentration (1-3mm). DNA polymerases from the green algae, however, display a pH optimum between 7.5 and 8.5 and an optimum Mg(2+) concentration of 8mm. With all algae, a higher polymerase activity was obtained with denatured salmon sperm DNA as template than with native DNA. All four deoxyribonucleoside 5'-triphosphates must be present for full activity of the polymerases. 2. With one exception, the deoxyribonuclease activities in the preparations, measured under conditions of the DNA polymerase assay, are low compared with corresponding preparations from Escherichia coli. Chlamydomonas extracts contain a high deoxyribonuclease activity. 3. After purification on columns of DEAE-cellulose, the polymerase activity was linear over a wide range of protein concentrations, except for Chlamydomonas preparations, where the observed deviation from linearity was probably attributable to the high nuclease activity. 4. DNA polymerases from all these algae bind strongly to DNA-cellulose; 6-40-fold purifications of the enzyme were obtained by chromatography on columns of DNA-cellulose. 5. The partially purified polymerases of Euglena and Anacystis are heat-labile but become much more heat-stable when tested in the presence of DNA.
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PMID:The activity of deoxyribonucleic acid polymerase in some species of algae. 462 75

Two group I introns (CpSSU.1 and CpSSU.2) that each potentially encode a protein with two copies of the LAGLI-DADG motif were identified in the Chlamydomonas pallidostigmatica chloroplast small subunit rRNA gene. They both belong to subgroup IA3 and represent novel insertion positions in this gene (sites 508 and 793 in the Escherichia coli 16S rRNA). The proteins encoded by the two introns were synthesized in vitro and tested for their ability to cleave the homing site of their respective introns. Only the CpSSU.1-encoded protein (I-CpaII) was found to display specific DNA endonuclease activity. At 0.1 mM MgCl2, I-CpaII nicks only the bottom (transcribed) DNA strand, but at concentrations ranging from 0.5 to 5.0 mM, it cleaves both DNA strands (leaving a 4 nucleotide single-stranded extension with 3'-OH overhangs) while preferentially nicking the bottom strand. The rate of cleavage of the top strand increases with increasing concentration of MgCl2. The preliminary data derived from these endonuclease assays suggest that the mode of DNA cleavage by I-CpaII is directed by the availability of Mg2+ and the affinity of different binding sites for this cation.
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PMID:The site-specific DNA endonuclease encoded by a group I intron in the Chlamydomonas pallidostigmatica chloroplast small subunit rRNA gene introduces a single-strand break at low concentrations of Mg2+. 763 Jul 30


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